[Jonathan Weber, FRCP, FRCPath, FmedSci, is Head, Section of Infectious Diseases, Division of Medicine, Imperial College School of Medicine, St. Mary's Hospital, London, United Kingdom.]
19 October 2000
Also available at http://www.medscape.com/viewarticle/408272
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The Royal Society's discussion meeting on the origins of HIV and the AIDS epidemic was organized by Simon Wain-Hobson of the Pasteur Institute and Robin Weiss of the Windeyer Institute, University College Hospital Medical School, London. The third organizer of the meeting, the evolutionary biologist Bill Hamilton, died earlier in 2000 after contracting malaria during an expedition to the Congo to capture chimpanzees for simian immunodeficiency virus (SIV) testing. Hamilton was interested in testing the hypothesis -- brought to widespread attention by the publication of journalist Edward Hooper's book The River in 1999 -- that the emergence of the HIV could be attributed to the inadvertent transfer of SIVcpz into humans by an oral polio vaccine (OPV) developed in monolayer cultures of kidney cells from infected chimpanzees. However, the Royal Society meeting was more than just a debate between OPV developers and proponents of the OPV transfer theory. It also reviewed much of the evidence for the theory that SIVcpz entered humans as a result of routine interspecies contact -- the so-called natural transfer or "cut hunter" theory -- and offered a compelling case for why the origins of HIV are of more than academic interest. The meeting was also a vindication of the courage of the Royal Society to call a public discussion meeting to bring together a scientific panel with investigative journalists and other commentators on the origin of AIDS.
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The meeting opened with an overview of cross-species infections, given by Professor Albert Osterhaus of Erasmus University, Rotterdam, The Netherlands. He noted that new pathogens are constantly emerging from animal reservoirs to challenge animal and human populations. Of these, HIV is undoubtedly the most famous zoonosis of the latter half of the 20th century, although the Spanish influenza epidemic of 1918-1919 claimed more human lives than all the conflicts of the 20th century combined. Osterhaus showed that cross-species epidemics are an annual event. The decimation of North Sea seals by a canine paramyxovirus and the appearance of related viruses in dolphins and porpoises are recent examples. All human infectious diseases ultimately have an animal origin, and natural transfer of these infections is a common event in animal populations. Human transfers are seen regularly with influenza A, an avian virus initially, transferred to humans generally through pigs. However, the recent Hong Kong chicken influenza outbreak in humans showed that the threat posed by zoonotic viruses is ever-present, and continuous vigilance will be required.
Dr. Kevin de Cock of the Centers for Disease Control and Prevention, Atlanta, Georgia, gave a rigorous overview of the epidemiological principles that should underlie any discussion of the origins of HIV. He described how The River proposes an ecologic association between contaminated OPV and the emergence of HIV and noted that the hypothesis relies on data that carry with them a high degree of bias and chance in their assembly. For example, on the basis of 38 cases of AIDS identified in the medical literature before 1980 and 58 HIV-1 isolates from stored serum samples, Hooper posits an association between OPV vaccination sites and the earliest manifestation of AIDS. However, these sources are inevitably subject to selection bias, as these cases had unusual characteristics, which caused them to come to the attention of subsequent investigators. Much valuable sera was destroyed in Kinshasa during the 1980s, for example, and Kevin de Cock pointed out that case reports in the medical literature are predominantly the product of academic medical centers, most likely to be accessed by Europeans. In addition, the AIDS cases identified by Hooper and other historians, such as the assiduous Mirko Grmek, lack confirmatory serologic data in most cases. OPV and early AIDS cases might be noncausally associated due to other confounding factors, such as proximity to major transport routes.
De Cock also criticized attempts to analyze SIV distribution among chimpanzees in Central Africa as epidemiologically irrelevant to understanding what took place in the past, just as current HIV prevalence tells us nothing about the date of HIV's emergence in a population. While Kinshasa has yielded evidence of HIV infection as far back as 1959, prevalence has remained stable at around 6% in pregnant women for the past decade, whereas HIV prevalence has climbed to considerably higher levels in southern Africa despite much later introduction of HIV. De Cock argued that the interplay of risk factors is a key determinant in the evolution of the HIV epidemic in a specific locale: male circumcision, history of genital herpes, and female sexual debut before the age of 16 were factors significantly associated with a higher HIV prevalence in a comparative prevalence study of 4 cities in Cameroon, East Africa, and southern Africa.
Dr. Leopold Zekeng of the Cameroon Laboratoire de Sante Hygiene Mobile reviewed HIV and SIV infections in Cameroon and the theory that monkey-hunting Pygmy tribes in the region might have been the vectors for the transfer of HIV from chimps to the wider population. Although Cameroon provides the richest diversity of HIV groups in the world, playing host to infections with HIV-1 groups M, N and O, the more recently identified groups are very rare, with a stable prevalence of HIV-1 group O (2% to 3%) and only 5 identified cases of group N infection among 6500 individuals sampled. Within group M, subtype A and A/G recombinants are predominant. Zekeng and colleagues investigated HIV prevalence among 2 Pygmy tribes and found that it was lower than among the predominant Bantu and strongly associated with contact with urban centers, suggesting that the theory that Pygmy hunters have acted as a bridge for HIV into the human population is probably unfounded.
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What is the primate reservoir from which HIV emerged? Dr. Preston Marx of Tulane Regional Primate Research Center, Covington, Louisiana, pointed out that unless we understand how SIV turned into HIV we run the risk of new zoonoses; understanding which primates might present a future reservoir, were current variants of HIV ever to be controlled with vaccines, is thus a crucial area of HIV prevention research rather than simply an academic dispute. For example, smallpox has been eradicated by vaccinia immunization; however, a related virus, monkeypox, can infect humans and could theoretically re-emerge as a new zoonosis. We might be wise to continue vaccination for this reason.
Professor Beatrice Hahn of the University of Alabama, Birmingham, presented new data on her team's efforts to characterize the species reservoir of SIVcpz and its geographical range. SIVcpz is agreed to be the simian ancestor of HIV-1 group M, and Hahn has reported the reservoir to reside in Pan troglodytes troglodytes, the Central African chimpanzee that is largely confined to Gabon, Congo Brazzaville, Cameroon, and a small range of northern Congo (now the Democratic Republic of Congo).
Assessing SIVcpz prevalence in the wild is difficult because captured animals are invariably infants. Prevalence is very low among infant chimps, suggesting that maternal transmission is inefficient and rare. Hahn and colleagues developed fecal and urine antibody and HIV-1 RNA tests to measure prevalence, which were validated against Western Blot tests in 3 captive SIV-infected chimps.
The group then analyzed urine and fecal samples from Pan troglodytes troglodytes in Côte d'Ivoire and found no evidence of SIVcpz in West Africa. However, 1 infected chimp has been identified in East Africa at an unidentified location, and Dr. Hahn said that she expected to begin analysis of a viral sequence shortly. Western Blot analysis of the antibodies recovered shows a different reactivity from SIVcpz isolates recovered from chimps captured in Central and West Africa.
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Presentations by Betty Korber of Los Alamos National Laboratory in New Mexico and Anne-Mieke Vandamme of the Rega Institute in Belgium both dated the last common ancestor for all HIV group M subtypes at around 1930. If this ancestor was a human immunodeficiency virus, this would be the very latest date at which transfer from chimps to humans might have occurred. The Rega Institute group estimates that the transfer could have occurred anywhere between 1590 and 1760, with 1675 the most likely date.
The technique used by both groups involved estimating the speed of molecular change within chosen viral genetic sequences, by comparing a variety of sequences from different dates. The Rega group removed from their calculations all sites within a sequence that did not conform to the molecular clock, a dating approach already verified in individuals with a known hepatitis C infection date in 1977. A group of Irish patients were exposed to anti-D immunoglobulin at this time, and HCV isolates from infected individuals were analyzed in 1998 in a blinded fashion to validate the technique. The group found that the more nonconforming sites they removed from the sequence, the more accurate their dating.
The Los Alamos group did not assume a stable rate of evolution, and used the earliest known HIV isolate (collected in Kinshasa in 1959) in comparison with other later isolates to verify their method. They examined the V3/V5 regions of the HIV envelope from 197 sequences gathered in Zaire in April 1997 and compared prior sequences from the same region to establish evolution rates. They established a likely date of 1940, with confidence intervals extending from 1871 to 1955.
Professor Paul Sharp's group at Nottingham University has analyzed whether there was an acceleration of genetic change in passages through new hosts, as represented by recombination events in the lineage. They hypothesized that evidence of recombination events would increase in periods where the viral subtype encountered new hosts more frequently, and that these would be detectable in the structure of the phylogenetic tree of particular subtypes. They analyzed the gag evolutionary tree and found that recent HIV-1 group M branches have a much higher ratio of changes that suggest adaptation to a new host, compared with older branches of the tree, implying that HIV-1 group M variants may have evolved slowly, sequestered in a manner similar to HIV-2, until they began to be transmitted more frequently and encountered many new hosts.
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Dr. Tom Burr, a statistician from Los Alamos National Laboratory, addressed the question of whether it is genetically plausible that the HIV epidemic could have been generated from a single cross-species transfer. Does the fact that there are so many distinct clades of HIV represent many introductions from a common SIV source, or a single introduction that diversified widely as it spread?
Dr. Burr presented the results of an analysis of phylogenetic trees of HIV and feline immunodeficiency virus (FIV) which demonstrated some of the peculiarities of HIV's evolution. The genetic tree of FIV appears to branch down a central line, but the HIV-1 group M tree radiates from a central node, suggesting a single transfer event. The Los Alamos group used a software package called Treevolve (developed at the University of Oxford) to simulate the genealogy of a sample given a variety of values for rates of partner change, new HIV cases per year, and time to AIDS death, based on UNAIDS data gathered between 1980 and 1998. They found 4 possible patterns in these models, only 1 of which was consistent with their HIV phylogenetic tree. The distribution of subtypes in the phylogenetic tree that was created when they modeled an epidemic spread of HIV from a single time point was the only one that resembled the phylogenetic tree currently considered to be the most accurate representation of HIV evolution. This suggested to Dr. Burr that a point source of the HIV epidemic was plausible, which might support the OPV theory. However, as discussed below, even if multiple introductions of SIV occurred in humans, it is equally plausible that only one lineage might result in an epidemic, with other introductions petering out spontaneously.
Three groups of HIV-1 exist today: group M, group O, and group N. While group M is pandemic, groups O and N are not and remain closely restricted to a single location. HIV-2 consists of 1 clear group with a separate path of descent from the form of SIV found in sooty mangabeys in West Africa. This genetic tree suggests 4 separate transfers to humans, a small number of events given the potentially frequent contact with chimps in Central Africa, and if the dating of chimp to human virus transfer back to the 18th century or even earlier is accurate. This discrepancy is one that requires further investigation and, of course, further refinement of the techniques of molecular epidemiology. However, as noted above, many zoonotic introductions might have occurred with dead-end infections, with only a few introductions leading to established human transmissions.
In the opinion of Daniel Low-Beer, an epidemiologist from the University of Oxford, a star-like geographic pattern of infection would need to have been established early in the epidemic; otherwise, chance extinction events in one village could have eliminated the new virus from the human population quickly. He estimates that at least 60% of SIV transfers were dead-end infections of this sort, which may have flared and died in remote locations.
Sir Robert May of University of Oxford, one of the world's foremost mathematical biologists, suggested that it was plausible for many viral transfers to have remained confined in locations where local custom did not encourage mixing between populations in different villages. By his calculations, if the virus spread equally within a village and outside a village, this would result in the extinction of the virus within a village, and a ripple effect that would result in lower and lower prevalence as the virus traveled from its original "hearth." The virus would establish a foothold in a new village only to die out because of low rates of partner change and would be transferred out to other villages if inhabitants took partners in other places, and so on. This effect would result in an initial peak followed by a slow decline over several decades -- estimated at between 30 and 40 years if the average number of individuals infected by each case was only slightly more than 1 -- before the virus moved into geographically concentrated populations with high rates of sexual partner change, as found in the urban conditions of Kinshasa in the 1970s, for example. Very small changes in sexual activity at the high partner end of the spectrum will have a dramatic effect on the basic reproductive rate and may be difficult to detect by standard epidemiological techniques, such as questionnaires and interviews. This theoretical model might explain the difficulty in finding SIV-infected humans representing transfer events in cross-sectional studies, while also explaining how an epidemic might arise as low-level rural infection, barely exceeding the basic reproductive rate (R0), meets a dense social network in the urban environment.
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If SIV crossed the species barrier in several places and then began to spread slowly, where are the most likely places for this to have occurred?
A review of subtype diversity by Martine Peeters found that the greatest diversity -- indicative of the greatest duration of opportunities for recombination with other viruses -- is found in the vicinity of Kinshasa, and that Congo generally shows the greatest degree of subtype diversity of any region in Africa. But does this necessarily make Congo the ancestral hearth of HIV or just the crossroads at which a number of viruses were able to come together?
Francine McCutchan reviewed current knowledge about circulating recombinant forms of HIV and the evidence for recombination events in the ancestry of different HIV subtypes. East and South Africa have a low level of recombinants, but in West and Central Africa the dominant subtypes, including recombinant forms and the "pure" subtypes, show evidence of common recombinant break points that indicate a common recombinant ancestor. This in turn would indicate a greater degree of opportunities for recombination events, both in terms of duration of infection in the region and rates of partner change. Whether the higher frequency of recombinants is a marker for the length of time HIV-1 been circulating in the region is debatable.
As sequence information accumulates and phylogenetic dating becomes more sophisticated, it may become easier to answer, but at present any assertions on the location of first transfer are highly speculative.
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The Royal Society meeting was originally envisaged as a forum for analysis of the hypothesis that an experimental OPV called CHAT was contaminated by SIVcpz, that the contaminated vaccine was fed to upwards of 1 million people in various parts of the Congo, Burundi, and Rwanda between 1957 and 1959, and that this vaccine transferred a low level of SIV into the human population.
The hypothesis has been raised by several investigators over the past 10 years, and has been elaborated most extensively by Edward Hooper, who was invited to present the evidence that supports his case. He was then answered by Dr. Stanley Plotkin and Dr. Hilary Koprowski, developers of the vaccine. In the interests of brevity, Hooper's allegations and the researchers' rebuttals are dealt with point by point.
Hooper argues that the attenuation of this particular polio virus was carried out by passaging it in a cell monolayer culture derived from chimpanzee kidney cells, rather than the macaque kidney cells used routinely for polio vaccine research at that time, and still used preferentially today. Plotkin says chimpanzee kidneys were never used because they were not a suitable substrate. It should be noted that there is no published or unpublished report from this era suggesting that chimp kidney cells were used for propagating polio virus.
Hooper argues that the development of the vaccine was carried out either at laboratories in the Congo or at the Wistar Institute in Philadelphia, Pennsylvania, and the Rega Institute in Antwerp, Belgium, and that there is inconsistent evidence about what substrate was used. All those directly involved in the development of CHAT say that the vaccine stock was propagated on macaque kidney cell monolayers and that chimp kidney cells were never used. Hooper's allegation that vaccine stocks were grown and purified in the Congo is not supported by testimony from any of the scientists involved.
Hooper says that he has signed statements from 2 individuals who worked at a chimp-holding facility called Lindi and a research station at Bujumbura in Rwanda that chimp kidneys were removed and shipped to other locations. In the case of Lindi camp, Hooper's witness said that staff members told him that the shipments of kidney were destined for the United States. However, this witness was indirectly reporting what had been said and had no first hand information.
Koprowski stated categorically that the chimps at Lindi were used only for challenge experiments to demonstrate the safety of CHAT OPV, and that the only organ shipments that took place from Lindi were the livers of primates used in hepatitis B experiments at the camp. Hooper has also suggested that a Polish researcher called Jezierski experimented with chimp kidney cells as a growth medium at a research station called Gabu Nioka in the north of Congo. Dr. Jan Destrymer says that chimp kidneys were not used at Gabu Nioka for vaccine development, based on evidence provided by Jezierski in papers published at the time.
Hooper claims that different batches of CHAT were prepared in different places but given the same number, so that the batch 10a/11 held by the Wistar Institute and supposedly used in the Congo may have been passaged further in the Congo in chimp kidney cells or another batch may have been manufactured in the Congo and given the same batch number. Plotkin describes this supposition as absurd and showed his original laboratory records for the batch, which show that lot 10a/11 was used in Sweden and New Jersey, as well as in Congo.
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Investigators of the OPV theory have repeatedly called for the remaining lots of CHAT vaccine to be tested for SIV and HIV and for mitochondrial DNA to be analyzed to establish which species' cells were used to propagate the vaccine. Two lots of vaccine are known to have survived into the 1990s: one lot (10a/11) is held by the Wistar Institute, while the other was held by the Karolinska Institute in Stockholm and was tested for HIV a few years ago. The Wistar Institute recently released its stock of CHAT vaccine to be tested under the supervision of an independent panel headed by Dr. Claudio Basilico of New York University School of Medicine. Basilico reported the results of those tests at the Royal Society meeting.
Using Roche Amplicor polymerase chain reaction, the collaborative group responsible for testing found no evidence of HIV-1 RNA or SIV RNA in any of the samples. Most important, mitochondrial DNA was detected in these samples, despite their age, and was shown in independent laboratories on blinded samples to be macaque in origin. No chimpanzee DNA was detected in any sample.
The HIV-1 assay had a limit of detection of 100 copies/mL, while the SIV assay had a detection limit of 1000 copies/mL. Philippe Lena of the Pasteur Institute in Paris used more sensitive DNA tests to examine whether SIV could persist in macaque kidney cells when they were prepared in the fashion employed by vaccine developers. He reported that kidney tissue from a macaque with plasma SIV RNA of 122 x10^6 copies/mL contained 571 SIV DNA copies/mcg of tissue, and replication-competent virus, which persisted in CEMX174 co-culture after 6 passages. Lena concluded that while the quantities of DNA were possibly too small to be infectious in this case, macrophages, which could harbor retrovirus infection, were detectable in the kidney cell monolayer, "whizzing about all over the place" as Robin Weiss put it in his summary of the meeting.
However, John Garrett of the UK's National Institute of Biological Standards and Control (NIBSC) assessed the viability of HIV and SIV in monkey kidney monolayers exposed to trypsin, an agent used in the preparation of the cells used to culture polio vaccine in macaque kidneys. HIV and SIV did not replicate in the presence of trypsin in the NIBSC experiments. Edward Hooper has suggested that trypsin was not routinely used in the late 1950s, but John Beale argued that it would not have been possible to achieve the polio virus titers reported by Stanley Plotkin unless trypsinization took place. Beale showed that the process used to prepare polio virus vaccine from macaque kidney cell monolayers would lead to a 10 to 12 log10 reduction in SIV infectivity. Indeed, polio virus vaccine continued to be prepared from African green monkey kidney cell monolayers in the 1970s and 1980s. Although these monkeys harbor an SIVagm virus, the process for OPV has not lead to SIV contamination of OPV.
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Preston Marx of Tulane Regional Primate Research Center noted that although the OPV hypothesis may in his view be fatally wounded, that does not exclude other iatrogenic possibilities. Marx presented his research into the potential role of nonsterile injecting equipment not only in spreading HIV, but in turning it into an aggressive human virus in the first place.
Marx began to be interested in this possibility after looking for human HIV-2 infection in West Africa that might be associated with sooty mangabey contact. These primates are the hosts of SIVsm, the ancestor of HIV-2, and Marx was surprised to find little evidence of SIVsm infection among the population. Only 2 of 9509 individuals tested in Sierra Leone were HIV-2-infected, despite no difficulty in finding SIVsm-infected mangabeys kept as household pets. Might HIV-2 be a relatively nonpathogenic infection, also difficult to transmit onward due to extremely low viral titer? Cohort evidence certainly suggests this is so, and that HIV-2 can incubate for upwards of 40 years without causing disease.
Had something occurred to amplify the virulence of HIV-1 after its transfer from chimpanzees? It is known that SIV becomes more aggressive if passaged through a new host, especially if the index case is experiencing primary infection. Might HIV-1 not do the same? And what would provide the most efficient means of passaging HIV through multiple hosts? This hypothesis is attractive at explaining how the adaptation of SIVcpz to a new host (humans) might have occurred rapidly, through sequential passage mediated by hypodermic needles.
Marx points to the massive increase in the use of antibiotics and injections in Africa during the 1950s. Before the Second World War, syringe use was limited because they were expensive and made from glass. Plastic syringes were introduced in 1959, when their cost dropped up to 100-fold, and the use of penicillin and choloroquine became more common in Africa. Re-use of syringes was so commonplace as to be unremarkable and continues to this day. Marx pointed out that "if we are correct about this, ...we will continue to get new strains. It could wreck vaccine research."
Charles Gilks of Liverpool University School of Tropical Medicine noted that the extremely high prevalence of hepatitis C in Egypt is directly attributable to injectable treatments used to combat schistosomiasis in mass campaigns during the 1950s. He also pointed out that human/chimpanzee contact accelerated hugely in the 20th century driven by the imperatives of medical and scientific research. Chimps were used in malaria experiments because it was noted that they carried 3 of 4 of the malaria parasite species that affected humans. Doctors are known to have injected themselves with blood from malarial chimps in the 1930s, and subsequent experiments included the injection of chimp blood into 20 individuals in Antwerp and prisoners in the United States. It was feared at the time that if malaria was eradicated by measures such as DDT spraying in the developing world, chimps might serve as a reservoir for renewed human exposure.
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All human viral infections were initially zoonotic in origin. Animals will always provide a reservoir for viruses that could threaten human populations in the future. The OPV, which has successfully controlled poliomyelitis worldwide, was propagated in monkey kidney cells potentially contaminated with SIV; yet by good fortune, the process failed to lead to human contamination. Although there is thankfully no evidence that OPV is the cause of the AIDS epidemic, we can see that this was a near-miss. As Professor Robin Weiss observed, there are many examples of transfer of other retroviruses from animals to humans, including iatrogenic transfer of retroviruses. The Royal Society meeting was a welcome opportunity to review concerns about the potential role of medical science in causing or amplifying the HIV epidemic. We must be constantly vigilant about our ability to inadvertently transmit infections through human interventions, as the BSE epidemic and threat of vCJD must constantly remind us. There are now compelling data to refute OPV as the cause of AIDS. However, there is still a myriad of currently unknown viruses in animal populations on land, sea, and air with the potential to cause human disease. It is a sobering thought by how little we may have avoided a major epidemic.